Vector-mediated gene transfer engenders long-lived neutralizing activity and protection against SIV infection in monkeys

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1 Vector-mediated gene transfer engenders long-lived neutralizing activity and protection against SIV infection in monkeys Philip R Johnson, Bruce C Schnepp, Jianchao Zhang, Mary J Connell, Sean M Greene, Eloisa Yuste, Ronald C Desrosiers & K Reed Clark Nature America, Inc. All rights reserved. The key to an effective HIV vaccine is development of an immunogen that elicits persisting antibodies with broad neutralizing activity against field strains of the virus. Unfortunately, very little progress has been made in finding or designing such immunogens. Using the simian immunodeficiency virus (SIV) model, we have taken a markedly different approach: delivery to muscle of an adeno-associated virus gene transfer vector expressing antibodies or antibody-like immunoadhesins having predetermined SIV specificity. With this approach, SIV-specific molecules are endogenously synthesized in myofibers and passively distributed to the circulatory system. Using such an approach in monkeys, we have now generated long-lasting neutralizing activity in serum and have observed complete protection against intravenous challenge with virulent SIV. In essence, this strategy bypasses the adaptive immune system and holds considerable promise as a unique approach to an effective HIV vaccine. Development of an HIV vaccine is proving to be a daunting task. In the years since the discovery of HIV, hundreds of vaccine candidates have been vetted in a variety of animal models. Many have also been tested in early-phase human clinical trials with mostly disappointing results. Two vaccine approaches, each targeting a different arm of the adaptive immune response, have been evaluated in large efficacy trials. Both failed to protect vaccine recipients from infection, and neither diminished viral replication after infection. Although there are other candidates in the pipeline, it seems unlikely that a noteworthy breakthrough is imminent. These sobering observations underscore the tremendous hurdles that must be overcome to develop an effective HIV vaccine. Foremost among these hurdles is the inability to induce antibodies that neutralize a wide array of HIV field isolates. Such antibodies are rarely found in the sera of long-term infected humans,, and only a handful of broadly neutralizing human monoclonal antibodies have been isolated. Therefore, one can conclude that broadly neutralizing antibodies are both relatively rare and difficult to elicit, even after acute and chronic natural infection. It seems unlikely that vaccine developers will improve upon natural responses with contrived immunogens, at least in the near term. Passive immunization schemes using neutralizing antibodies have protected monkeys from SIV or simian-human immunodeficiency virus (SHIV) challenge infections. Unfortunately, an injection of antibodies every few weeks is neither practical nor cost effective as a large-scale human vaccine approach. Some years ago, it was shown that sustained delivery of antibodies could be achieved in mice by implanting collagen-encapsulated T fibroblasts that had been transduced ex vivo with a retroviral vector carrying an antibody gene. HIV-specific antibodies that seeped out of the implant decreased viral burden in HIV- infected, humanized immunodeficient mice. Our interest in adeno-associated virus (AAV) vectors caused us to consider such vectors as a means to deliver HIV-specific antibodies directly to muscle, thereby avoiding ex vivo manipulations. In this scheme, the antibody gene of choice is packaged into an AAV vector, which is then delivered by direct intramuscular injection. Thereafter, antibody molecules are endogenously synthesized in myofibers and passively distributed to the circulatory system. In a proof-of-concept experiment, mice injected with an AAV vector carrying the gene encoding IgGb produced authentic, biologically active IgGb that was detected in mouse sera for over months. These data, although encouraging, were generated in mice, which is an artificial model system that is sometimes difficult to translate to higher-order primates. Moreover, it was unclear whether the serum levels of neutralizing activity generated would translate to protection from a challenge infection. To more rigorously test the basic concept of antibody gene transfer as an approach to immunization, we turned to the well characterized SIV model system in monkeys. Here we describe the adaptation of the original antibody gene transfer concept to macaques and also detail the derivation of macaque-specific designer molecules (immunoadhesins) that neutralize SIV and afford protection from SIV challenge infection. The Children s Hospital of Philadelphia and the University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, USA. The Research Institute at Nationwide Children s Hospital, Columbus, Ohio, USA. New England Primate Research Center and Harvard Medical School, Southborough, Massachusetts, USA. Correspondence should be addressed to P.R.J. (johnsonphi@chop.edu). Received February; accepted April; published online May ; doi:./nm. NATURE MEDICINE VOLUME [ NUMBER [ AUGUST

2 L S L V Linker C H C H Hinge C H C H C H C H C H C H N because of its unique composition, despite the fact that it did not neutralize as potently as L or L. We individually cloned expression cassettes for L and L between AAV inverted terminal repeats designed to produce selfcomplementary genomes. Such genomes have been shown to direct more efficient in vitro and in vivo transduction when compared to traditional single-stranded DNA AAV vectors. N is a traditional single-stranded DNA genome because we made it before the selfcomplementary vector was available to us. We packaged all three recombinant genomes into AAV serotype capsids by standard techniques. Nature America, Inc. All rights reserved. N CD (D and D) C H C H Figure Schematic representation of immunoadhesin constructs. For molecules derived from macaque Fabs (L, S, L and V), and domains were joined by a synthetic linker. Rhesus CD (domains and (D and D)) was cloned as described in the Methods online. Antigen-binding domains were attached to the Fc fragment of a rhesus IgG molecule. RESULTS SIV immunoadhesins In pilot experiments in mice (J.Z., B.C.S., P.R.J. and K.R.C., unpublished data), we showed that immunoadhesins (defined as chimeric, antibody-like molecules that combine the functional domain of a binding protein with immunoglobulin constant domains) were superior to single-chain (scfv) or whole-antibody (IgG) molecules with respect to achievable steady-state serum concentrations. Our constructs followed a formula whereby an antiviral moiety was fused to an IgG Fc domain (Fig. ). To generate SIV-specific immunoadhesins with native macaque sequences, we obtained previously characterized SIV gp-specific Fab molecular clones that had been derived directly from SIV-infected macaques. These Fab molecules were shown to neutralize several defined stocks of SIV in vitro, thereby affording us the opportunity to test in vivo neutralizing activity after gene transfer to macaques. For these experiments, we chose two Fabs for further modification: -h and -h (ref. ). Both Fabs had potent in vitro neutralizing activity against SIVmac, a macrophage-tropic derivative of SIVmac (refs. ). Consequently, we chose SIVmac as our challenge strain. Although SIVmac has not routinely been used in SIV vaccine experiments, it was ideally suited for our purposes because we were able to match antibody reagents with a pathogenic SIV strain. In the end, the SIVmac challenge stock was highly infectious and pathogenic for rhesus monkeys (see below). We joined the variable heavy ( ) and variable light ( )chains from the Fabs via a linker to create an scfv and then joined the scfv to arhesusiggfcfragment(fig. ). Because the linear order of the and moieties was arbitrary, we made both orientations for each Fab. We also included a construct based on the rhesus CD domains and, modeled after CD-g fusion proteins. When we transfected the plasmid into cells, the expressed proteins were secreted into the cell culture medium as disulfide-linked homodimers (data not shown). They all bound SIV gp in a standard ELISA, and each construct showed substantial in vitro neutralizing activity against SIVmac (Fig. ). We subsequently chose constructs L and L (representing Fabs -h and -h, respectively) for testing in macaques. We also chose to test Immunization by in vivo gene transfer We immunized nine rhesus macaques with AAV vectors carrying immunoadhesin constructs: three received L, three received L and three received N. We administered each vector at time by intramuscular injection ( vector genomes), and we periodically collected serum from each macaque and tested it for protein abundance and biologic activity. Immunoadhesin concentrations in the L vector recipients reached mg ml by weeks after immunization, which was the time of challenge (Fig. a). These concentrations corresponded well with measured serum neutralization titers on the day of challenge ranging from in, to in, (Fig. b). Two L-immunized monkeys (C and C) had a prechallenge course similar to the L-immunized monkeys. Serum concentrations of the immunoadhesin reached mg ml and mg ml, respectively, by the day of challenge (Fig. a), which corresponded to measured serum neutralization titers of in, and in, (Table ). The third L recipient (C) was different. The L immunoadhesin was present at weeks after immunization ( mg ml ), but was undetectable by weeks after immunization (Fig. b). This rise and fall was reflected in serum neutralization titers that were in, at weeks and o inat weeks after immunization (Table ). This decline reflected the appearance of L-specific antibodies in this macaque (details below). The three macaques immunized with N had much lower serum concentrations of immunoadhesin on the day of challenge ( mgml ; Fig. c) when compared to L-immunized monkeys. Serum neutralizing titers were likewise lower in these macaques, ranging from in to in (Table ). The lower titers are probably due to the fact that N is a single-stranded DNA vector, resulting in less efficient vector transduction. SIV challenge Four weeks after immunization, we gave all nine macaques an intravenous injection of SIVmac. In addition, at that time we SEAP activity (%), L S L V N..... Concentration (µg ml ) Figure Neutralization of SIV in vitro. In vitro neutralizing activity against SVmac of the five immunoadhesins depicted in Figure. All five showed substantial % neutralization at o mg ml. The half-maximal inhibitory concentration (mg ml ) for each was L (.), S (.), L (.), V (.) and N (.). SEAP, secreted alkaline phosphatase. VOLUME [ NUMBER [ AUGUST NATURE MEDICINE

3 Nature America, Inc. All rights reserved. a Serum concentration (µg ml ) c Serum concentration (µg ml ) d injected three unimmunized control macaques with the same dose of the same challenge stock. Sixteen weeks later, we inoculated three more unimmunized control macaques with the same dose of the same challenge stock (for a total of six naive control macaques). Viral loads after challenge are shown in Figure. The challenge virus infected all six unimmunized control macaques, with peak plasma viral loads ranging between. to RNA copies ml (Fig. d). Only a single control macaque (D) suppressed viral replication to below copies ml in the postacute phase of infection. In fact, we had to kill four of the six macaques between and weeks after infection, owing to AIDSrelated complications. With death as an endpoint, the immunized macaques were significantly protected (zero of nine died) relative to the unimmunized controls (four of six died; P ¼.). In contrast to the control monkeys, six of the nine immunized macaques remained uninfected after challenge, as judged by a lack of SIV RNA in plasma (Fig. a c) and lack of antibodies to Gag (Supplementary Fig. online). All three of the L macaques were protected from infection (compared to unimmunized controls, P ¼.), while two of three were protected in the N group (P ¼.), and one of three in the L group (P ¼.). Humoral responses after SIV challenge As expected, all six naive control monkeys had SIV-specific antibody responses after infection. All developed antibodies to gp (Fig. d) and Gag (Supplementary Fig. ). There was also a concomitant rise in serum neutralizing activity. By weeks after challenge, neutralization titers in the three naive controls ranged from in, to in, (Table ). We did not test the three control macaques (C, D and D) infected at weeks after the first group for neutralizing antibodies before or after challenge. Likewise, the three immunized macaques that became infected after challenge (C, C and D) also developed antibodies to gp and Gag (Supplementary Fig. ). Shortly after challenge, each had sizable increases in gp-specific antibodies detectable by ELISA that were clearly distinguishable from those generated by AAV vector mediated gene transfer (Fig. b,c). These higher levels also correlated with increases in serum b Serum concentration (µg ml ) Serum concentration (µg ml ) C D, D, C C D,, C D D C D D C C C Figure Serum concentration of immunoadhesins or antibodies after gene transfer and SIV challenge. (a d) Serum concentrations of immunoadhesins or antibodies in L immunized macaques (a), L-immunized macaques (b), N-immunized macaques (c) and unimmunized controls infected at two different times ( month and. months) (d). Sera from unimmunized and immunized macaques were tested over time by a SIV gp ELISA for immunoadhesins (or antibodies in infected macaques) after immunization and challenge infection (see Methods online). The day of challenge was month after immunization. In b, the reactivity to gp for monkeys C and C was off scale (owing to SIV infection) and peaked at, and, mg ml, respectively. In c, the reactivity to gp for monkey D was off scale (owing to SIV infection) and peaked at, mg ml. The pink shaded areas in a c represent the time period after immunization and before challenge. neutralization titers. By weeks after challenge, titers in these monkeys ranged from in, to in, (Table ). The six immunized macaques that remained uninfected after challenge had a distinctly different profile. All three monkeys that received L were protected from challenge infection and had serum neutralizing activity on the day of challenge that was maintained through weeks after challenge (Table ). Serum L concentrations gradually rose to a peak at about months after immunization and ultimately plateaued between and months at mg ml (Fig. a). L recipient C was also protected from infection and had a pattern of immunoadhesin abundance (Fig. b) and neutralizing activity (Table ) similar to the L monkeys. This general pattern of expression was consistent with the biology of AAV vectors and has been shown for other transgene products in mice, dogs and monkeys. Moreover, unlike antibodies that arise in response to SIV infection, but like the authentic immunoadhesins, the gp-binding and neutralizing activity in the sera of these monkeys at months after immunization was completely sensitive to heat treatment at for min (data not shown). Two N monkeys were also protected (C and D), even though serum neutralization titers on the day of challenge were much lower than in the other four protected monkeys (Table ). As with the other protected monkeys, serum concentrations of N were maintained through months, albeit at much smaller concentrations ( mg ml ). Table Serum neutralizing activity against SVmac before and Time (weeks) after immunization Vaccine Macaque (DOC) None C o o o o, D o o o o, D o o o o, L C o,,, C o,,,, D o,,, L C o,,, C o, o o, C o,,,, N C o D o, D o Titers shown are reciprocal serum dilutions representing % neutralization. The day of challenge (DOC) was at week. NATURE MEDICINE VOLUME [ NUMBER [ AUGUST

4 Nature America, Inc. All rights reserved. a (log per ml) c (log per ml) after challenge C C D C D D after challenge Correlates of protection Variations in protection among immunized macaques allowed us the opportunity to search for potential correlates of protection. A clue came from macaque C, who had an early peak and then a rapid decline in serum L, such that by the day of challenge, neutralizing activity in the serum was below the threshold of detection (Table ). This scenario was similar to expression patterns observed in mice with transgene-specific antibody responses (J.Z., B.C.S., P.R.J. and K.R.C., unpublished data). To explore this possibility, we used purified L protein as the solid-phase antigen and tested C serum in a standard ELISA (Fig. a). The reactivity pattern showed a high-titered L-specific antibody response that was detectable by weeks after immunization, peaked at weeks and declined thereafter (Fig. a). These data are consistent with the peak and rapid decline of the L immunoadhesin in the serum of macaque C, and they also explain why the macaque became infected. We next tested serum from the rest of the immunized macaques in the same fashion (Fig. a). It was readily apparent that the other two immunized monkeys that became infected (C and D) also had immunoadhesin-specific antibody responses, albeit at much lower levels than C (Fig. a). None of the protected monkeys showed immunoadhesin-specific responses (Fig. a). In contrast to macaque C, macaques C and D possessed in vitro serum neutralizing activity on the day of challenge (Table ). In fact, macaque C had the highest serum neutralization titer of any of the macaques ( in,) on the day of challenge (Table ). These data prompted us to examine the specificity of the immunoadhesin-specific responses. Antibodies from each macaque had a distinct specificity (Fig. b). Serum from C (L recipient) reacted with purified L and L, but not N, suggesting that these antibodies were raised against the framework sequence in the scfv domain of L and L. Serum from C (L recipient) reacted with all three immunoadhesins, suggesting that these antibodies were raised against the Fc fragment that each chimeric protein had in common. b (log per ml) d (log per ml) after challenge after challenge a A nm C C C C C D D C D D C L C Figure Detection of SIV in plasma after challenge. (a d) SIV viral loads after challenge infection of L-immunized macaques (a), L-immunized macaques (b), N-immunized macaques (c) and unimmunized controls (d). Plasma samples from unimmunized and immunized macaques were tested after challenge infection for SIV RNA genomes (viral load). The day of challenge was months (and. months for the second set of unimmunized control macaques). Monkeys C, D, D and D from the unimmunized control group were killed between and weeks after infection, owing to AIDS-related complications. Finally, serum from D (N recipient) reacted only with N, indicating specificity for the CD moiety that is unique to N. DISCUSSION In the experiments described here, we used gene transfer technology to effectively bypass the adaptive immune system to generate long-lived, protective anti-siv biological activity in the sera of otherwise naive monkeys. Six of nine immunized monkeys were protected against infection by the SIV challenge, and all nine were protected from AIDS, whereas all six of the controls became infected and two-thirds (four of six) died over the course of the experiment. Much of the success in these experiments can be attributed to AAV gene transfer technology. AAV vectors have an established record of high-efficiency gene transfer in a variety of model systems,.when delivered to post-mitotic organs such as muscle, brain and liver, AAV vector genomes take on the form of intranuclear high molecular weight episomal concatamers that direct transgene expression for extended periods of time. Our previous work demonstrated the feasibility of this approach in mice for in vivo HIV neutralization, and other investigators have adapted AAV (and other vector systems) for antibody gene delivery for a variety of purposes.herewe showed that in monkeys a single intramuscular injection of an AAV vector could direct long-term ( year) continuous expression of a biologically active protein. In addition, the serum immunoadhesin concentrations achieved with the self-complementary (doublestranded DNA) AAV vectors (L and L) were far superior to those observed with the traditional single-stranded DNA vector (N). Three key caveats emerged from our data. First, transgene immunogenicity seemed to be a major correlate of protection that must be better understood. We tested three different immunoadhesins and observed immunoadhesin-specific responses that ran the gamut from C D N D C Time after immunization weeks weeks weeks weeks weeks weeks weeks C L D b A nm C (L) C (L) D (N) L L N Figure Immunoadhesin antibodies in immunized monkeys. (a) Reactivity of serum ( in dilution) collected over time from each immunized macaque for immunoadhesins, as tested by ELISA. For example, sera from L recipients C, C and C were reacted against purified L protein (as indicated on the x axis). The dashed line indicates the limit of sensitivity. The inset shows sera from macaque C at a in, dilution. (b) Reactivity of sera from the three indicated macaques for the respective purified proteins (as indicated on the x axis). As expected, each macaque serum reacted with the homologous immunoadhesin. C reacted against L (homologous) and L (heterologous). The dashed line indicates the limit of sensitivity. VOLUME [ NUMBER [ AUGUST NATURE MEDICINE

5 Nature America, Inc. All rights reserved. undetectable (L) to almost completely incapacitating (L). It is tempting to speculate, but remains unproven, whether immunoadhesins expressed by in vivo transduction might behave like exogenously administered recombinant DNA derived proteins (for example, monoclonal antibodies or chimeric molecules such as etanercept) whose safety profiles can be readily established. Moreover, it should be possible to lower the potential for immunogenicity by specific residue modifications,. Notably, none of the three macaques who showed immunoadhesin-specific antibody responses has shown clinical signs or symptoms of disease. A second caveat is that traditional in vitro neutralization assays did not seem to faithfully represent in vivo activity. Although macaques without measurable neutralizing activity on the day of challenge were all infected, the opposite was not true; the presence of in vitro neutralizing activity, even at high titer, did not guarantee protection. Macaque C showed an immunoadhesin-specific antibody response, directed at the Fc domain of the chimeric protein, that did not inhibit in vitro neutralizing activity. In fact, this macaque had the highest neutralizing titer of all of the immunized macaques on the day of challenge (Table ). These data suggest that immunoglobulin effector functions not measured in standard in vitro assays (such as Fc activity) might be crucial for in vivo neutralizing activity. Notably, our data seem to confirm recent work in which the Fc effector function of a neutralizing monoclonal antibody was inactivated by mutation, and the in vivo protective effect of the antibody was blunted. These observations suggest that optimizing antibody functions over and above antigen binding might be of benefit in future iterations of antiviral transgenes. A final caveat is that we used the intravenous route for the SIV challenge. Because most new HIV infections occur across a mucosal surface, it will be crucial to show protection against a mucosal challenge. However, there is cause for optimism. Several studies have shown that traditional systemic passive immunization with antibody preparations can protect against an SIV or SHIV mucosal challenge. Moreover, there is ample evidence to suggest that systemic immunization with a nonreplicating immunogen can protect from natural infection at a mucosal surface. A recent example is the vaccine against human papillomavirus, which is a nonreplicating virus-like particle that, when given intramuscularly, elicits antibodies that protect against infection ofthefemalegenitaltract. Notably, the macaques in the current study immunized by gene transfer maintained high levels of circulating antibodies that probably permeated most body tissues and mucosal surfaces. Although considerable hurdles remain, the HIV immunization approach outlined here seems to be a viable alternative to more traditional strategies. To ultimately succeed, more and better neutralizing monoclonal antibodies against primary HIV isolates that can be tested in gene transfer experiments will be needed. Also, more nonantibody inhibitors such as CD and its derivatives should be developed in anticipation of combinatorial approaches that target multiple steps along the HIV entry pathway. And, finally, as we have shown here, the SIV-monkey model can be used to investigate a variety of variations on this theme. METHODS Methods and any associated references are available in the online version of the paper at Note: Supplementary information is available on the Nature Medicine website. ACKNOWLEDGMENTS We thank A. Hessell and D. Burton (The Scripps Research Institute) for providing the SIV Fab molecular clones, D. McCarty (The Research Institute at Nationwide Children s Hospital) for the self-complementary AAV vector genome, R. Doms (University of Pennsylvania) for purified SIVmac gp, J. Bixby and E. Mackenzie for technical assistance and M. Piatek and J. Lifson for SIV viral load data. We also thank J. Hoxie and S. Douglas for helpful comments on the manuscript. Funding for this work was provided by grants from the US National Institutes of Health National Institute of Allergy and Infectious Diseases Division of AIDS (P.R.J. and R.C.D.), National Institutes of Health National Center for Research Resources (R.C.D.) and support from The Children s Hospital of Philadelphia. 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7 Nature America, Inc. All rights reserved. ONLINE METHODS Simian immunodeficiency virus specific immunoadhesin gene constructs. We synthesized DNA (Geneart) with optimized codons. We used SIV Fab -h to derive L and S, and we used Fab -h to derive L and V. We joined the variable domains by a amino acid glycine-serine (GS) linker, and we placed a synthetic signal peptide for optimized secretion at the end. The Fc fragment was rhesus IgG cloned from lymphocyte RNA. We placed each construct between a cytomegalovirus promoter (similar to one previously published ) and a synthetic polyadenylation signal. N contained the rhesus CD signal sequence followed by the D and D domains of rhesus CD. Recombinant proteins. We transfected (Superfect, Qiagen) HeLa cells (American Type Culture Collection) with plasmid, and we purified proteins from the medium using protein-a (Nunc International). We quantified purified proteins by ELISA, using purified rhesus IgG as a standard (Bethyl Laboratories). Adeno-associated viral vectors. We produced and purified all vectors as previously described,,. Titers ranged between and vector genomes per ml. Monkeys and immunization. We purchased rhesus macaques of Indian origin from Covance and housed them in the vivarium at the Research Institute at Nationwide Children s Hospital in accordance with standards set forth by the American Association for Accreditation of Laboratory Animal Care. All macaques tested negative for antibodies to SIV, simian type D retrovirus and simian T-cell lymphotropic virus type. Their weights at the time of immunization ranged from. kg to. kg. We gave each immunized macaque vector genomes divided into four equal portions (. ml each), delivered by four separate (two in each quadriceps) deep intramuscular injections. Simian immunodeficiency virus neutralization assay. We tested purified proteins or macaque sera for in vitro neutralization activity with the previously described secreted alkaline phosphatase assay. Immunoadhesin concentrations. We measured L, L and N concentrations in serum by using SIV gp to coat ELISA plates ( ng per well) in PBS buffer at C overnight. We decanted the antigen and blocked the wells with % BSA in PBST (PBS +.% Tween-) for h with constant shaking at C. After washing with PBST, we incubated the wells with ml ofdiluted serum (or L, L or N standards) for h at C. After washing, we added horseradish peroxidase conjugated secondary goat antibody to human IgG-Fc HRP (Bethyl Laboratories; in,) to each well ( ml) for min at C. After four more washes, we added tetramethylbenzidine substrate ( ml, Pierce) for min at C. After we terminated the reaction with N H SO, we read the plates at an absorbance at nm on a Molecular Devices microplate spectrophotometer. We quantified the results by extrapolation from the standard curve with software from Molecular Devices (coefficient of linearity Z.). The assay sensitivity was. ng ml for L and L and ngml for N. Transgene-specific antibody responses. We coated plates with purified N, L or L purified proteins in PBS buffer ( ng per well) overnight at C and processed them as described above. We added mouse antibody to human IgG ( in dilution; Sigma-Aldrich) in blocking buffer to the wells and incubated them at C for min. We chose an IgG isotype specific secondary antibody to avoid cross-reactivity with the IgG immunoadhesins. After washing, we incubated the wells with ml (in,dilution)ofa rabbit mouse IgG-Fc specific horseradish peroxidase conjugate (Sigma- Aldrich) for min. We performed TMB substrate development as described above. To detect cross-reactivity in specific monkey serum samples, we processed alternative coating antigens in an identical manner as that used for the cognate coating antigen. Simian immunodeficiency virus challenge. We generated infectious SIVmac by transfecting full-length proviral DNA into T cells (American Type Culture Collection) by the calcium phosphate method (Promega). We measured virus concentrations in the supernatant by determining the concentration of p capsid protein using an antigen-capture assay according to the manufacturer s instructions (SIV p Antigen Capture Assay, Advanced Bioscience Laboratories). The virus stock used in this study contained ng ml of p. We diluted the virus stock in, in sterile PBS and injected ml intravenously. We estimated that ml of the diluted stock contained approximately macaque infectious doses by comparison of its infectivity in cell culture to that of other SIV stocks. We determined viral loads in plasma with a quantitative real-time RT-PCR assay as previously described. Statistical analyses. Because of small sample sizes, we used Fisher s exact probability test (two-tailed) to examine the differences between groups.. Barash, S., Wang, W. & Shi, Y. Human secretory signal peptide description by hidden Markov model and generation of a strong artificial signal peptide for secreted protein expression. Biochem. Biophys. Res. Commun., ().. Ostedgaard, L.S. et al. A shortened adeno-associated virus expression cassette for CFTR gene transfer to cystic fibrosis airway epithelia. Proc. Natl. Acad. Sci. USA, ().. Levitt, N., Briggs, D., Gil, A. & Proudfoot, N.J. Definition of an efficient synthetic poly(a) site. Genes Dev., ().. Clark, K.R., Liu, X., McGrath, J.P. & Johnson, P.R. Highly purified recombinant adenoassociated virus vectors are biologically active and free of detectable helper and wildtype viruses. Hum. Gene Ther., ().. Lifson, J.D. et al. Role of CD + lymphocytes in control of simian immunodeficiency virus infection and resistance to rechallenge after transient early antiretroviral treatment. J. Virol., (). NATURE MEDICINE doi:./nm.